INDUCIBLE REPAIR OF NEAR-UV RADIATION DAMAGE

这项研究计划的总体目标是通过使用 操纵子融合，参与一个独特的修复系统，由 近紫外线B(NUV-B；290-320 nm)和近紫外线A(NUV-1； 320-400 nm)辐射损伤，并确定哪些条件 调控这些基因的表达。自然紫外线的生物响应 辐射包括明显不同于 远紫外光(FUV；200-290 nm)。大肠杆菌利用的机制 应对NUV应激可能与其他全球氧化应激有关 管理系统；NUV应激蛋白可能对生物体至关重要 存活和正常细胞功能不可或缺的物质类似于 热休克蛋白。有证据表明，这些应激反应 原核细胞和真核细胞的活性可能是相同的。 额外的NUV辐射，特别是NUV-B(290-320 nm)可能会增加 作为臭氧消耗的结果；这将增加 皮肤黑色素瘤。日光灯会增加人体对NUV-A的暴露 辐射。这项研究最初将集中在识别修复 低注量的广谱近紫外辐射诱导的操纵子 利用转座子Tn5-lac将lac Z表达融合到外源 推动者。这项研究的工作假设是Katf和/或毛皮 是一个可诱导的监管网络的成员，该网络控制 自然紫外线相关特定基因亚集的协同表达 保护和修复。其他可能的候选者是APAH、OXR、SOXR 和oxoR。具体目标：(1)分离Tn5乳胶操纵子融合 在低注量率(225W/m2)下由NUV诱导，以及(2) 确定影响转录调控的条件 这些歌剧中。研究设计和方法：修复手术将被 通过利用P1：Tn5 Lac构建随机操纵子融合来鉴定。 Tn5转座子将LacZ的表达融合到外源启动子和 只与Tn5以外的启动子进行转录融合。 传感器将被复制镀在X-GAL上，并经过循环 无紫外线照射15分钟，然后无无紫外线照射30分钟，连续72小时。TN5紫胶 显示NUV诱导的操纵子融合将被转导到菌株 含有已知影响对NUV敏感性的基因突变 (Kate，katG，DAM，Pola，Soda，SodB，xthA)，以及在含有 已知的氧化应激调节基因的突变(oxR， SoxR、oxoR、APAH、FUR、KATF)，然后筛选对 转录调控。辐射率、通量和比 波长(NUV-A与NUV-B)也将进行测试。特别强调 将集中在铁摄取调节子(FUR)和 Atf基因对乳酸菌转录调控的研究。其他可能的情况 调节性突变也将使用Tn10构建。 所有Tn5-lac操纵子融合的转录调控和前景 调控突变体将通过测量水解度来详细研究 LacZ基因产物β-半乳糖苷酶在液体培养中的ONPG 在人工NUV或天然条件下连续搅拌时 阳光。他们将在各种不同的通量比率下进行筛选 和不同的波长范围(NUV-B和NUV-A)来确定 诱导或抑制参与调控的基因的条件， 修复和保护细胞免受NUV胁迫。

The overall goal of this research program is to identify genes, using operon fusions, that are involved in a unique repair system induced by near-ultraviolet-B (NUV-B; 290-320 nm) and near-ultraviolet-A (NUV-1; 320-400 nm) radiation damage, and to identify those conditions which regulate the expression of these genes. The biological response of NUV irradiation include effects that are distinctly different from those of far-ultraviolet light (FUV; 200-290 nm). Mechanisms utilized by E. coli to cope with NUV stress may link up with other global oxidative stress management systems; NUV-stress proteins may be vital for an organisms survival and integral to normal cell functioning in a similar matter to heat shock proteins. Evidence indicates that these stress response activities may be the same in both prokaryotic to eukaryotic cells. Additional NUV radiation, particularly NUV-B (290-320 nm) may increase as a result of ozone depletion; this will elevate the incidence of cutaneous melanoma. Tanning lamps increase human exposure to NUV-A radiation. This study will initially concentrate on identifying repair operons induced by low fluence levels of broad-spectrum NUV irradiation utilizing Tn5-lac, a transposon that fuses lac Z expression to exogenous promoters. The working hypothesis for this study is that katF and/or fur are members of an inducible, regulatory network that controls the coordinated expression of a specific subset of genes involved in NUV protection and repair. Other possible candidates are apaH, oxyR, soxR and oxoR. Specific aims: (1) Isolation of Tn5 lac operon fusions that are induced by NUV at low fluence rates (225 W/m2) and (2) the identification of conditions which affect the transcriptional regulation of these operons. Research Design and Methods: Repair operons will be identified by constructing random operon fusions utilizing P1:Tn5 lac. Tn5 is transposon that fuses lacZ expression to exogenous promoters and makes only transcriptional fusions to promoters outside Tn5. Transductants will be replica plated on X-gal and subjected to cycles of 15 minutes NUV followed by 30 minutes without NUV for 72 hours. Tn5 lac operon fusions showing induction by NUV will be transduced to strains containing mutations in genes known to influence sensitivity to NUV (katE, katG, dam, polA, sodA, sodB, xthA), and in strains containing mutations in genes known to be regulators for oxidative stress (oxyR, soxR, oxoR, apaH, fur, katF), then screened for effects on transcriptional regulation. Radiation rate, fluence and specific wavelengths (NUV-A vs. NUV-B) will also be tested. Particular emphasis will be focused on the influence of the ferric uptake regulon (fur) and the katF genes on transcriptional regulation of lac. Other possible regulatory mutations will also be constructed using Tn10. Transcriptional regulation of all Tn5 lac operon fusions and promising regulatory mutants will be studied in detail by measuring hydrolysis of ONPG by the lac Z gene product, beta-galactosidase in liquid cultures while continuously stirred under artificial NUV or under natural sunlight. They will be screened under a wide variety of fluence rates and different wavelength ranges (NUV-B and NUV-A) to determine those conditions that induce or suppress genes involved in regulating, repairing and protecting cells from NUV stress.